1. Field of the Invention
The present invention relates generally to the biochemical pharmacology of ophthalmologic agents. More specifically, the present invention relates to synthetic complementary peptides and ophthalmologic uses thereof.
2. Description of the Related Art
Alkali-injury of the eye provokes an acute inflammatory reaction, largely composed of polymorphonuclear leukocytes (PMNs), which are responsible for corneal ulcerations and perforations.1-3 N-acetyl-PGP and N-methyl-PGP, neutrophilic chemoattractants released during direct alkaline hydrolysis of corneal proteins, are the initial triggers for polymorphonuclear leukocyte invasion into the alkali-injured cornea.4-6 The specific activity of N-acetyl-PGP is greater than the methylated tripeptide.4 
Recognition that N-acetyl-PGP is an important mediator in this disease has opened a therapeutic window of opportunity. Early inhibition of this chemoattractant in an alkali-injured eye might reduce or eliminate the first neutrophilic influx. Minimizing the number of neutrophils initially penetrating into the damaged cornea would limit the production of secondary inflammatory mediators, such as leukotriene B4, hence reducing the additional recruitment of polymorphonuclear leukocytes. Exclusion of neutrophils from the alkali-injured cornea is the key to decreasing or eliminating corneal ulceration. It is therefore of paramount importance to search for lead compounds which can inhibit this chemoattractant.
One approach to the development of a lead inhibitory compound can be found in the molecular recognition theory.7 This concept posits that a fundamental requirement for biological reactions is that proteinaceous molecules recognize one another in a genetically defined manner. Blalock and Smith8 proposed a novel approach to molecular recognition which has succeeded in predicting the interactions of proteinaceous molecules with high frequency. This method, based on the development of complementary peptides specified by ligand antisense RNA, has proven useful in designing interactive peptides, isolating receptors, and producing anti-receptor and anti-idiotypic antibodies.9,10 
Thus, the prior art is deficient in synthetic complementary peptides to treat ophthalmologic disorders. The present invention fulfills this long-standing need and desire in the art.
The present invention demonstrates an application of the molecular recognition theory, which is the generation of therapeutic agents that may be used to treat disease. Using this approach, a series of complementary peptides for the pro-gly-pro (SEQ ID NO:1) sequence were designed, synthesized, and tested as antagonists of the PMN chemoattractant, N-acetyl-PGP.
In an embodiment of the present invention, there is provided a pharmaceutical composition for ophthalmologic uses. Specifically, such composition is a complementary peptide which comprises complementary sequences to proline-glycine-proline (PGP). Generally, the complementary sequences are designed based on the possible coding triplet for proline and glycine and on the hydropathic value of the two amino acids. Enhancement of the potency of the complementary sequence was achieved with a multimerization process. The resulting molecule can be divided into 4 specific subunits, connected by amide bonds with different functions: 1) recognition subunit 2) core multimerizing subunit 3) spacer subunit and 4) R N-terminal subunit.
The recognition subunit: the complementary sequence to Pro-Gly-Pro, this subunit is responsible for the interaction with the chemoattractant. The recognition subunit is present as a single unit in the monomer, is repeated twice in the dimer, 4 times in the tetramer and 8 times in the octamer. It is defined by the sequence all-L Arg-Thr-Arg and by the sequence all-L Xxx-Thr-Arg (Xxx=the 20 natural amino acids), and by all-D Arg-Thr-Arg and all-D Xxx-Thr-Arg (Xxx=the 20 natural amino acids).
The core multimerizing subunit, absent from the linear monomers, is characterized by a branching di-amino amino acid (lysine, di-amino propionic acid, di-amino butyric acid) connected to a single alanine, where both amino groups are involved in an amide bond. The function of the core is to determine the number of recognition units in the molecule and to control the relative spatial distribution of the recognition subunits. The core also represents the connection point to the resin during Solid Phase Peptide Synthesis. The octameric core is defined by the formula all-L (((B)2B)2)B-Ala, the tetramer by all-L (B)2B-Ala and the dimer by all-L B-Ala (where B=lysine, di-amino propionic acid and di-amino butyric acid). The core was also obtained with all-D amino acids with the same generic formulas.
The spacers represent the connection point between the core and the recognition subunits and determines the relative spatial distribution of the recognition subunits. It can be constituted by a di-glycine. The di-glycine could be substituted by a single amino acid with the formula: NH2[CH2]nxe2x80x94COOH [n=2[3-amino propionic acid];3;4;5;6; or 7[8-amino caprylic acid]]
R-terminal subunit: A free amino terminal group on each recognition subunit is not necessary for the subunit function. This group can be functionalized by an R molecule to modify the pharmaco-dynamic properties of the molecule and to produce a more constrained molecule. The R can be H3Cxe2x80x94(CH2)nxe2x80x94CO with n=0 (acetyl), n=4 (caproyl) and n=14 (palmitoleyl). R can also be the amino acid cysteine. In the case of the tetrameric peptide the sulfur groups could be used for the formation of an intra molecular di-sulfide bridge, generating a constrained bi-cyclic molecule.
In another embodiment of the present invention, there is provided a method of inhibiting polymorphonuclear leukocyte polarization, chemotaxis and infiltration into tissue activated by a neutrophil chemoattractant in an individual by administering the pharmaceutical composition of the present invention to the individual. Preferably, the neutrophil chemoattractant is selected from the group consisting of N-acetyl-PGP, N-acetyl-PGX, N-methyl-PGX, N-methyl-PGP and small peptide chemoattractants containing proline and glycine. Still preferably, the pharmaceutical composition is administered at a concentration range of from about 1 xcexcM to about 100 mM, depending on the peptide.
In still another embodiment of the present invention, there is provided a method of treating an eye disease in a n individual by administering the claimed pharmaceutical composition. Preferably, the pharmaceutical composition is administered at a concentration range of from about 1 xcexcM to about 100 mM, depending on the peptide. Representative eye diseases which can be treated using this method of the present invention include alkali-injured eye, chemically injured eye or inflammatory diseases of the eye which are well known to those having ordinary skill in this art.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.